Pigment

ABSTRACT

This invention relates to a new pigment in feed for salmonides, a new feed comprising this pigment and use of this pigment. The pigment comprises a diester of astaxanthin prepared with an omega-3 fatty acid and/or a short chain carboxylic acid. By this invention a pigment for feed to salmonides that is more stable and biologically more effective than free astaxanthin and commercially available astaxanthin product, is provided.

This application is a 371 application of PCT/NO00/00129 filed Apr. 17,2000.

This invention relates to a new pigment in feed for salmonides, a newfeed comprising this pigment and use of the pigment.

In feed for farmed salmon and trout pigment has to be added to obtainthe desired colour of the fish flesh. The pigment mostly used isastaxanthin which corresponds to the pigment which is available in feedfor wild salmonides. Also other pigments like for instance cantaxanthin,might be used. Such pigments are very unstable with regard to exposureto air and temperature as well as light. The pigments are therefore to agreat extent degraded during feed processing and storage. These pigmentsare all carotenoids. This feed is mostly prepared from raw material notcontaining significant amounts of astaxanthin (i.e. white fish). Farmedsalmon and trout is fed industrially manufactured feed where pigment isadded.

Commercially available astaxanthin products are furthermore veryexpensive and their biological retention is very low. Astaxanthin is asmentioned above a rather unstable compound, which of course is adrawback. The low stability of astaxanthin is due to oxidation.Commercial pigment products are formulated in order to avoid or reduceoxidation. One typical formulation for astaxanthin is with gelatine andstarch. The formulations used are often, however, not optimal withrespect to biological availability of the pigment, and a new way ofsolving the stability problem, combining a high degree of stability withimproved biological availability would be of great economical benefit tothe aquaculture industry. A more stable pigment Is thus highly desiredas this would give possibilities for making a formulation more optimalwith regard to biological availability and consequently possibilitiesfor considerably economic saving.

Thus it is a desire in the aquaculture industry to find more stable andbiologically effective pigments useful in production of feed forsalmonides.

The different salmonid species differ in their ability to utilisedietary carotenoid. Rainbow trout (Oncorhynchus mykiss) has been foundto utilize the pigment in the feed more effectively than Atlantic salmon(Salmo salar) and sea trout (S. trutta).

Rainbow trout can also accumulate higher amounts of carotenoids in theflesh than Atlantic salmon and sea trout, but less than sockeye salmon(Oncorhynchus nerka) (Storebakken, T. and Ho, N. K., Aquaculture, vol.100, (1992), p. 209).

In salmon, dietary astaxanthin and canthaxanthin are deposited moreefficiently in flesh than in skin, which is in contrast to the rainbowtrout (Schiedt, K. et al., Pure & Appl. Chem. 57 (1985) 685-692).

Synthetically produced astaxanthin is normally present in unesterifiedform (i.e. diol). This is also the form assumed that the pigment isconverted to in the intestine before it is absorbed by the fish (O. J.Torrissen et al., Reviews in Aquatic Sciences, vol. 1, (1989) pp.209-225). In nature astaxanthin is often present as diester.

Simpson, K. L. and Kamata, T., Proc. World Symp. on Finfish Nutr. andFishfeed Technology, Hamburg. Jun. 20-23, 1978. Vol II. Berlin 1979, pp.415-424, reported a study for pigmentation of rainbow trout comparingastaxanthin, astaxanthin ester and astacene. Astaxanthin, astaxanthinester and astacene were extracted from shrimp coagulum. The pigmentswere dissolved in herring oil and added to the trout diet. Whenanalysing the fish, no distinction was made between flesh and skin. Thefish fed the diet consisting of astaxanthin ester contained much higherlevels of total astaxanthin than others. This indicated that astaxanthinester was more effective for the pigmentation of rainbow trout. However,on the same symposium Torrissen, O. and Braekkan, O. R. (Proc. WorldSymp. on Finfish Nutr. and Fishfeed Technology, Hamburg. Jun. 20-23,1978. Vol II. Berlin 1979, pp 377-382) also demonstrated thatastaxanthin was incorporated into the flesh of rainbow trout. Theseauthors found that astaxanthin was more effectively incorporated inflesh than diesters and monoesters purified from the copepod, Calanusfinmarchicus.

According to O. J. Torrissen et al., Reviews in Aquatic Sciences, vol.1,(1989) pp. 209-225 (i.e.: Foss, P. et al., Aquaculture, vol. 65, (1987),p.293 and Storebakken, T. et al., Aquaculture, vol. 65, (1987), p. 279)synthetic astaxanthin diester (i.e. astaxanthin dipalmitate) seems to beabsorbed less easy than free astaxanthin both in rainbow trout, seatrout and Atlantic salmon.

In crustaceans, a relatively large part of the astaxanthin is present inester form. However, the pigment is more easily absorbed than whatshould be expected from the level of free astaxanthin. This is in O. J.Torrissen et al., Reviews in Aquatic Sciences, vol. 1, (1989) pp.209-225 tentatively explained from other not identified compounds incrustacea which might contribute to enhanced absorption.

To summarise: as astaxanthin absorption in the intestine of the fish isassumed to involve free astaxanthin (i.e. diol) it is so far mainlyconsidered that feeding with esters will give less biological absorptionthan feeding with free astaxanthin. This is supported by experimentswith astaxanthin dipalmitate.

It is known that astaxanthin present as diester is more stable than freeastaxanthin (Omara-Alwala, T. R. et al., J. Agric. Food Chem., vol. 33(1985), p. 260 and Arai, S. et al., Aquaculture, vol. 66 (1987), p.255.)

In the literature, dipalmitate is the predominant diester studied, andit is reported to give less pigmentation than the diol (Torrissen, O.and Braekkan, O. R.; Proc. World Symp. on Finfish Nutr. and FishfeedTechnology, Hamburg Jun. 20-23, 1978. Vol II. Berlin 1979, pp. 377-382,Storebakken, T. et al., Aquaculture, vol. 65 (1987), p. 279, Foss, P. etal., Aquaculture vol. 65 (1987), p. 293, Torrissen, O. J et al., CRCCrit. Rev. Aqua. Sci. vol. 1 (1989), p. 209. ). This is explained by alow degree of hydrolysis of the diester.

We have shown that by using a commercial lipase (Candida rugosa), after42 hours the dipalmitate hydrolysed to 40% free astaxanthin. Wesynthesised several other diesters in order to study whether thesehydrolysed faster than the dipalmitate. Example 1 shows that under thesame experimental conditions the diester with elaidic acid (trans-C18:1)hydrolysed to a higher degree (73%) while a short-chain carboxylic acid(C10:0) hydrolysed somewhat slower, and a diester prepared with aconcentrate of omega-3 fatty acids comprising approx. 50% EPA (all cisC20:5 n3) and approx. 35% DHA (all cis C22:6 n3) (in total more than 90%omega-3 fatty acids) (EPA+DHA) hydrolysed to free astaxanthin at lessthan half the rate of the dipalmitate.

To verify the hydrolysis data for these astaxanthin diesters obtained byusing the commercial lipase, another similar experiment was performed byusing enzyme isolated from the intestine of Atlantic salmon. Thisexperiment very surprisingly gave opposite data than the data obtainedin the experiment where the commercial lipase was used; i.e. the EPA+DHAdiester was hydrolysed quickest and the dielaidate and the dipalmitatewere hydrolysed slowest (see Example 2). Thus the inventors mostsurprisingly have found that if astaxanthin esterified with aconcentrate of omega-3 polyunsaturated fatty acids is hydrolysed byenzyme from the intestine of salmon, a surprisingly fast hydrolysis tofree astaxanthin is obtained compared to dipalmitate. Surprisingly, alsodiester with the short chain carboxylic acid (C10:0) was hydrolysed muchfaster than the dipalmitate, even though the rate of hydrolysis wassignificantly slower than hydrolysis of the EPA+DHA diester.

Based on these surprising data, the present inventors have found aastaxanthin EPA+DHA diester which most likely hydrolyses quickly to freeastaxanthin when fed to the salmon and thus is effective forpigmentation of salmon. In Example 3 it is shown by feeding experimentsthat this statement is correct. In a similar way, the inventors havefound that an astaxanthin diester with short chain carboxylic acids willbe suitable as a pigment with good stability and a high potential forpigmentation of salmonides.

Furthermore, it was surprisingly found by the inventors that the EPA+DHAdiester comprised acceptable stability properties for use inindustrially manufactured feed without formulation with gelatine orstarch (see Example 3). This was not expected, as omega-3polyunsaturated fatty acids are unstable compounds.

In Example 3 pigmentation of salmon with the EPA+DHA diester wascompared to pigmentation with a commercial free astaxanthin product(Carophyll Pink, Roche). Most surprisingly, it was found that thebioavailability as measured by astaxanthin uptake in salmon fillet was41% higher in the fish fed the EPA+DHA diester compared with the fishfed the commercial pigment. Thus, feeding with this astaxanthin diestersurprisingly gives enhanced biological absorption compared to freeastaxanthin.

It is a main object of the invention to provide a pigment for feed tosalmonides that is more stable and biologically effective than freeastaxanthin and commercial pigments for salmonides.

Another object of this invention is to provide a pigment which can beadded to the feed in less amounts than previously known pigments andstill give a satisfactory pigmentation of the flesh.

This and other objects are achieved by the attached claims.

The invention is further explained by examples.

EXAMPLE 1

Diesters of astaxanthin were prepared with the following carboxylicacids: elaidic acid (trans-C18:1), palmitic acid (C16:0), decanoic acid(C10:0) and a concentrate of omega-3 fatty acids comprisingapproximately 50% eicosapentaenoic acid (EPA) (all cis C20:5 n3) andapproximately 35% docosahexaenoic acid (DHA) (all cis C22:6 n3). Thedifferent diesters were firstly hydrolysed by a commercial lipase,Candida rugosa (Lipase AY, 30). This reaction gave the following resultsafter 42 hours at room temperature:

Diester Free astaxanthin (%) 18:1 73 16:0 40 10:0 30 EPA + DHA 18

These results show that the diester with polyunsaturated fatty acids ishydrolysed more slowly than corresponding diesters with saturated ormonounsaturated fatty acids.

Based on this experiment, the esterification with an omega-3 concentrategives slower hydrolysis to free astaxanthin, and one should thereforeexpect lower biological uptake than in feeding experiments withastaxanthin dipalmitate that have been described in the literature.

EXAMPLE 2

A similar experiment as Example 1 was performed with enzymes from salmonintestinal fluid. The intestinal fluid from salmon was collected asdescribed from cod (Lie, Ø. et al., Comp. Biochem. Physiol., 80B (3),(1985), pp. 447-450). The following results were obtained after 45 hoursat room temperature:

Diester Free astaxanthin (%) 18:1 1 16:0 1 10:0 6 EPA + DHA 25

Surprisingly, here a considerably higher conversion to free astaxanthinfrom the EPA+DHA-diester was obtained than from the other diesters. Thedielaidate and the dipalmitate were after 45 hours hydrolysed to adegree of just 1%, while the didecanoate was hydrolysed to a degree of6% and the EPA+DHA diester to a degree of 25%. These results were mostunexpectedly opposite of those from Example 1. It is hereby shown that ahigher biological uptake than what was achieved in the experiment withastaxanthin diesters that has been described in the literature, probablywill be obtained when feeding with an astaxanthin diester prepared witheither a concentrate of omega-3 fatty acids or a short chain carboxylicacid.

EXAMPLE 3

The EPA/DHA astaxanthin diester (ACD) from Example 1 and 2 (30 mg/kgcalculated as free (i.e. unesterified) astaxanthin) was added to thepellet by pilot plant production of fish fodder. Urea (2% weightrelative to fish oil) was added with water during extrudation. Theaddition of pigment and fat/oil was done by vacuum-coating of theextruded pellet. The EPA+DHA diester was added together with the fishoil. 200 mg/kg ascorbic acid had been added to the fish oil. Afterproduction, a weight average of 18.4 mg/kg was recovered in the pellet(18.1 mg/kg as diester, and approx. 0.3 mg/kg as hydrolysed ester). Thisshows that the EPA+DHA diester survives the production process insufficient degree to be utilised in practical feeding of fish. Severalbatches of this fodder composition was produced. Detailed analyticaldata are given in the calculation below.

Commercial astaxanthin (Carophyll Pink, Roche, 30 mg/kg calculated asfree astaxanthin) was added to fish fodder in the same manner as above.In this commercial product unesterified astaxanthin is finely dispersedin a stach-covered matrix of gelatine and carbohydrates. Ethoxyquin andascorbyl palmitate are added as antioxidants. The process and rawmaterials, including urea addition, were identical with what is givenabove, with the exception that the fish oil contained no ascorbic acid.After production, a weight average of 25.0 mg/kg free astaxanthin wasrecovered in the pellet. Several batches of this fodder composition wasproduced. Detailed analytical data are given in the calculation below.

The two fodder compositions were given to salmon. Initially, the averageweight of the fish was 70 g. In the calculations that follow, we do notinclude pigment content of fish before feeding started. The values wereidentical for both groups, and the numerical values are so small thatthey will not be significant for the conclusions.

After 8 months the average weight of 20 fish for each group was 1180 gfor the fish fed with astaxanthin diester, and 1153 g for the fish fedwith commercial pigment. The average astaxanthin content of the fishfillet was 3.34 mg/g for the fish fed with the diester and 3.23 mg/g forthe fish fed with commercial pigment. If we assume that the fish weightconsisted of 70% muscle, we have the following average values forastaxanthin content:

Fish fed with astaxanthin diester: 1.180 kg/fish×0.70×3.34 mg/kg=2.76 mg

Fish fed with commercial astaxanthin: 1.153 kg/fish×0.70×3.23 mg/kg=2.61 mg

The fish fed with astaxanthin diester had received the following fodder:

457.4 kg containing 18.4 mg/kg = 8416 mg  97.6 kg containing 17.1 mg/kg= 1669 mg  32.6 kg containing 17.9 mg/kg =  584 mg Sum = 10669 mg 

Divided by the average number of fish during the study, each fish hadreceived:

10669 mg/762 = 14.00 mg astaxanthin. This gives a bioavailability of19.7%. 100% × 2.76 mg/14.00 mg =

The fish fed with commercial astaxanthin had received the followingfodder:

463.2 kg containing 25.0 mg/kg = 11580 mg 101.0 kg containing 24.8 mg/kg=  2505 mg  32.5 kg containing 30.0 mg/kg =  976 mg Sum = 15061 mg

Divided by the average number of fish during the study, each fish hadreceived:

15601 mg/775 = 20.13 mg astaxanthin. This gives a bioavailability of 100× 2.61/20.13 = 13.0%.

Thus feeding with ACD surprisingly gives enhanced biological absorptioncompared to tree astaxanthin.

EXAMPLE 4 and 5 Isolation of Crude Enzyme Mixtures From Salmon and TroutIntestine

Tris buffer (0.25 M) solution was prepared by dissolving Tris (4.54 g;37.5 mmol) in 150 ml of distilled water. The pH of the buffer solutionwas adjusted to 8.0 by adding 2 M hydrochloric acid. All the hydrolysiswas done at room temperature so no special effort was necessary.

The astaxanthin diester was weighed accurately (˜40 mg, 33 mmol) alongwith 2.5 g of chremophor EL emulsifier (from BASF) into 100 mlErlenmeyer flask. The solution was stirred vigorously for 20 minutes(until homogeneous) and then 20 ml of the Tris buffer was added andstirred for additional 10 minutes. The solution was suction filtered andthe red filtrate was poured into 25 ml volumetric flask and diluted tothe mark with buffer. Concentration of all the solution was determinedspectrophotometically (492 nm) to be about 1 mg/ml, except for diacetatewhich was obtained in a lower concentration. All the solutions werestored in refrigerator wrapped in aluminium paper.

Isolation of the Crude Enzyme Mixture

Fresh salmon and rainbow trout, that had been fed in the last 12 hours,were obtained from local fish farms. At the laboratory the fresh fishwas cut open and the crude enzyme mixture extracted as follows: From thedigestion line there were a lot of narrow tubes surrounded by fattissue. Each of them was cut off and extracted by hand into a cooledcontainer. The viscous solution (−50 ml) was diluted to 100 ml with 0.25M Tris buffer and stirred for 30 minutes in an ice bath. Next step wascentrifugation at 11.000 rpm for 20 minutes at 4° C. (RC5C from DuPont). The water layer was extracted by pipette to another centrifugeglasses and the centrifugation repeated at 18.000 rpm for 30 minutes at4° C. The water layer was transferred to Erlenmeyer flask and quicklyfrozen by liquid nitrogen. Before starting the reaction the mixture wasthawed and purified by ultracentrifuge at 27.000 rpm for 45 minutes at4° C. (157.000 g at top). The lipase preparation was obtained as a clearyellow solution.

Hydrolysis

Into a 10 ml round bottom flask was added 2 ml of stock solution and 3ml of crude enzyme mixture from fish intestine. The flask was filledwith nitrogen before closing, wrapped in aluminium paper and stirred for48 hours. When finished, all the water was removed under reducedpressure (0.01 Torr) and redissolved in CH₂Cl₂. The solution wasfiltered through cotton wool plug and stored under nitrogen in a closedcontainer. Analytical TLC was used to monitor the progress of thereaction with 5% acetone in CH₂Cl₂ as eluent (Diol(R_(f)=0.0-0.1),Monoest. (R_(f)=0.3-0.5), Diest. (R_(f)=0.8-0.9)). To determine thedegree of hydrolysis all the samples were injected into analytical HPLC.[Eluent: 30% acetone in n-hexane. Column: Nucleosil 50-5 (2×30 cm)column. Flow rate: 0.15 ml/min. Detector: 470 nm (Diol R_(f)=0.4-0.5,Monoest. R_(f)=0.7, Diest. R_(f)=0.9-1.0)].

EXAMPLE 4

A similar experiment as in Example 2 was performed with enzymes fromsalmon intestinal fluid.

The diesters that were hydrolysed were prepared by esterification ofastaxanthin with the following carboxylic acids:

Acetic acid, C16:0 (palmitic acid), fatty acids from a fish oil rich inomega-3 fatty acids (approx. 18% EPA and 12% DHA, called K30 in thetables below), a concentrate of omega-3 fatty acids (approx. 30% EPA and20% DHA, called K55 in the tables below), the same concentrate of EPAand DHA as used in example 2 (approx. 50% EPA and 35% DHA, called ACD inthe tables below), purified EPA (more than 95% EPA, called EPA in thetables) and purified DHA (more than 90% DHA, called DHA in the tables).

Table 1 shows the degree of hydrolysis after 48 hours hydrolysis withcrude enzyme mixture from salmon intestine.

After 48 hours the diester of astaxanthin with palmitic acid (16:0) hadresulted in no free astaxanthin. However, it is known from the priorart, that this diester is hydrolysed in salmon and gives rise to anincreased level of pigment in the fish. Thus, this result indicates thatthe crude enzyme preparation was not in an optimal condition, presumablydue to proteases which may act to degrade the needed enzymes.

All other diesters with omega-3 fatty acids or a short chain carboxylicacid (acetat) gives rise to a higher hydrolysis of diester to diolcompared to the diester of astaxanthin with palmitic acid and willtherefore be expected to give rise to a higher uptake of pigment in thefish.

The highest degree of hydrolysis was observed with the diester ACD (9%).The relative amount of free astaxanthin was three times as high as thatfrom the diester of a lower concentrated fish oil (K30). The resultsfurther show that the diester of purified EPA is hydrolysed faster thanthe diester of DHA. However, surprisingly the concentrate of EPA and DHAcalled ACD is hydrolysed faster than purified EPA. The diester of aceticacid is hydrolysed to 7% free astaxanthin. However, it should be notedthat for this compound only 13% remains as diester when the reaction isended. This is much lower then for the other products that were tested,and indicates that if the reaction time had been prolonged, a highamount of free astaxanthin would have been obtained from this diester.Similarly, the only 27% of the diester of EPA remains unreacted.Accordingly, under in vivo conditions, it is expected that the diestersmay show even higher relative degree of hydrolysis to free astaxanthinthan what is demonstrated in table 1.

EXAMPLE 5

A similar example as Example 2 b was performed. However, for thisExample crude enzyme preparation from rainbow trout (Oncorhynchusmykiss) was used. The results are shown in Table 2. Table 2 demonstratesthat after 48 hours 71% of the diester of palmitic acid remainedunreacted. For the fish oil 37% was unreacted, while for ACD only 14%remained unreacted. As for salmon, we observe that EPA reacts fasterthan DHA (17 and 21% unreacted), however for rainbow trout we observethat an omega-3 concentrate like the ACD diester gives higher degree ofhydrolysis than the diester of purified EPA. Only very small amounts ofdiester of acetic acid (3%) remains, indicating that a diester ofastaxanthin with a short chain carboxylic acid will give very rapidhydrolysis in fish intestine.

These results are confirmed by looking into the relative amounts of freeastaxanthin in table 2. Here ACD shows the highest results, with 27%free astaxanthin, compared to only 11% for the diester of palmitic acid.

The results in Tables 1 and 2 below support the conclusions that anastaxanthin diester with a concentrate of omega-3 acids or anastaxanthin diester with short chain carboxylic acids will give higherbiological uptake in farmed fish than experiments with astaxanthindiesters that have been described in the literature.

TABLE 1 Ratio (%) between astaxanthin diester, monoester and diol, after48 hours hydrolysis with crude enzyme mixture from salmon intestine.Substrate Diesters (%) Monoesters (%) Diol (%) Acetate 13 80 7 16:0 8119 0 K30 61 36 3 K55 42 52 6 ACD 37 54 9 EPA 27 67 6 DHA 44 53 3

TABLE 2 Ratio (%) between astaxantin diester, monoester and diol, after48 hours hydrolysis with crude enzyme mixture form rainbow troutintestine. Substrate Diesters (%) Monoesters (%) Diol (%) Acetate 3 8314 16:0 71 18 11 K30 37 46 17 K55 25 52 23 ACD 14 59 27 EPA 17 58 25 DHA21 55 24

The concentrate of omega-3 fatty acids comprising approximately 50%eicosapentaenoic acid (EPA) and approximately 35% docosahexaenoic acid(DHA)(in total more than 90% omega-3 fatty acids) which is used inExample 3 to prepare the astaxanthin diester, is only one example andshall not be considered as limiting for the invention. A person skilledin the art will understand that other concentrates of omega-3 fattyacids, e.g. concentrates containing less than 90% omega-3 fatty acids,might be used for preparing a astaxanthin diester giving similar resultsas those shown in the examples. Astaxanthin diesters esterified withcarboxylic acids from marine oils containing omega-3 carboxylic acids orprepared from concentrates of omega-3 fatty acids comprising a totalamount of EPA and DHA from 18 to 100%, preferentially from 40 to 100%,are covered by this invention. More precisely astaxanthin diestersprepared from concentrates of omega-3 fatty acids comprising an amountof EPA from 8 to 98%, preferentially from 25 to 98%, and an amount ofDHA from 8 to 98%, preferentially from 15 to 98%, are covered by thisinvention.

Example 2 demonstrates that a diester with a short chain carboxylic acidwith enzymes from salmon intestines also gives faster hydrolysis to freeastaxanthin than a diester with palmitic acid. Thus such a diester willalso be suitable as a pigment with high stability and enhancedbiological uptake compared to commercial pigment formulations. InExample 2, we used decanoic acid. Other short chain carboxylic acidswill be carboxylic acids with a chain length C1-C12. For the personskilled in the art, it is evident that these acids can be both saturatedand unsaturated.

Furthermore, the person skilled in the art will see that the inventedastaxanthin diester might be stabilised in the same way as commercialastaxanthin pigments, i.e. by means of gelatine-matrix. The amount ofgelatine-matrix relative to the amount of pigment may be less for theinvented pigment than what is needed to stabilise commercial pigments.Alternatively or additionally the invented pigment might be stabilisedby antioxidants and/or by urea as described in our Norwegian patentapplication no. 19983050.

For a person skilled in the art it is obvious that a fatty acid diesterof astaxanthin is more fat soluble than free astaxanthin. This is ofcourse an advantage by the present invention because the pigment will beeasier to formulate in the fish feed which is rich in fat.

Astaxanthin can be produced from microbial sources. Typical examples ofpromising microbial astaxanthin sources are the yeast Phalli rhodozymaand the alga Haematococcus pluvialis. Phalli rhodozyma has astaxanthinin free form, while Haematococcus pluvialis astaxanthin is presentmainly (87%) as diesters (Johnson, E. A. and An, G-H. 1991. Astaxanthinfrom microbial sources. Cit. Rev. Biotechnol. 11(4):297. ) For theperson skilled in the art it is obvious that based on the presentinvention, increased bioavailibility of astaxanthin diester frommicrobial sources can be obtained by choosing microbial strain, and/orby feeding with suitable fatty acids, or precursors for such fattyacids, or by choosing other suitable fermentation conditions, so thatthe astaxanthin diester that is produced contain omega-3 fatty acidsand/or short chain carboxylic acids.

What is claimed is:
 1. A pigment comprising a diester of astaxanthinprepared with a carboxylic acid, wherein the carboxylic acid is aconcentrate of an omega-3 fatty acid comprising a total amount of allcis C20:5 n3 eicosapentaenoic acid (EPA) and/or all cis C22:6 n3docosahexaenoic acid (DHA) from 18 to 100%.
 2. A pigment according toclaim 1, wherein the omega-3 fatty acid comprises a total amount of allcis C20:5 n3 eicosapentaenoic acid (EPA) and/or all cis C22:6 n3docosahexaenoic acid (DHA) from 40 to 100%.
 3. A pigment according toclaim 1, wherein the omega-3 fatty acid comprises an amount of all cisC20:5 n3 eicosapentaenoic acid (EPA) from 8 to 98% and/or an amount ofall cis C22:6 n3 docosahexaenoic acid (DHA) from 8 to 98%.
 4. A pigmentaccording to claim 1, wherein the omega-3 fatty acid comprises an amountof all cis C20:5 n3 eicosapentaenoic acid (EPA) from 25 to 98% and/or anamount of all cis C22:6 n3 docosahexaenoic acid (DHA) from 15 to 98%. 5.A pigment according to claim 1, wherein the omega-3 fatty acid comprisesapproximately 50% all cis C20:5 n3 eicosapentaenoic acid (EPA) andapproximately 35% all cis C22:6 n3 docosahexaenoic acid (DHA).
 6. A feedfor salmonides comprising 25-70% by weight of proteins, 5-60% by weightof lipids, 0-40% by weight of carbohydrates, pigment and 0-15% by weightof one or more additional components, wherein the pigment is a diesterof astaxanthin prepared with a concentrate of an omega-3 fatty acidcomprising a total amount of all cis C20:5 n3 eicosapentaenoic acid(EPA) and/or all cis C22:6 n3 docosahexaenoic acid (DHA) from 18 to100%.
 7. A feed according to claim 6, wherein the additional componentsare selected from the group consisting of fillers, adhesives,preservatives, vitamins and minerals.
 8. A feed according to claim 6,wherein the omega-3 fatty acid comprises a total amount of all cis C20:5n3 eicosapentaenoic acid (EPA) and/or all cis C22:6 n3 docosahexaenoicacid (DHA) from 40 to 100%.
 9. A feed according to claim 6, wherein theomega-3 fatty acid comprises an amount of all cis C20:5 n3eicosapentaenoic acid (EPA) from 8 to 98% and/or an amount of all cisC22:6 n3 docosahexaenoic acid (DHA) from 8 to 98%.
 10. A feed accordingto claim 6, wherein the omega-3 fatty acid comprises an amount of allcis C20:5 n3 eicosapentaenoic acid (EPA) from 25 to 98% and/or an amountof all cis C22:6 n3 docosahexaenoic acid (DHA) from 15 to 98%.
 11. Afeed according to claim 6, wherein the omega-3 fatty acid comprisesapproximately 50% all cis C20:5 n3 eicosapentaenoic acid (EPA) andapproximately 35% all cis C22:6 n3 docosahexaenoic acid (DHA).
 12. Amethod for pigmentation of salmonides, which comprises administering adiester of astaxanthin prepared with an omega-3 fatty acid comprising atotal amount of all cis C20:5 n3 eicosapentaenoic acid (EPA) and/or allcis C22:6 n3 docosahexaenoic acid (DHA) from 18 to 100% to thesalmonides.
 13. A method of feeding salmonides which comprisesincorporating a diester of astaxanthin prepared with an omega-3 fattyacid comprising a total amount of all cis C20:5 n3 eicosapentaenoic acid(EPA) and/or all cis C22:6 n3 docosahexaenoic acid (DHA) from 18 to 100%as a pigment in feed for salmonides.